A global increase in invasive group A Streptococcus (GAS) disease (bacteremia, necrotizing fasciitis, and streptococcal toxic shock syndrome) that began in the 1980's has continued through the present decade and has focused attention on investigation of mechanisms of GAS pathogenesis. During the previous funding period, work in their laboratory and others further documented the central role of the hyaluronic acid capsular polysaccharide in GAS virulence in experimental models of local and systemic infection. These studies showed that the capsule interferes with the phagocytic killing, prevents internalization of GAS by epithelial cells, modulates adherence mediated by other GAS surface molecules, and acts as a ligand for attachment of GAS to CD44 on pharyngeal keratinocytes. Despite these other advances, it remains undefined how the capsule or other virulence determinants control the processes of tissue invasion and persistent colonization in the host. During the next funding period, their objectives are to define the role of the hyaluronic acid capsule in invasion of GAS from an epithelial surface to deep tissue, to characterize the effects of capsule on intracellular trafficking of GAS in epithelial keratinocytes, and to determine how regulation of capsule expression in vivo contributes to pathogenesis of GAS infection. The proposed experiments will make use of primary keratinocyte cultures and a model system simulating intact human skin in conjunction with confocal fluorescence microscopy in order to study the tissue and cell biology of GAS translocation through human skin, the phenomenon of persistence within cells, and the regulation of capsule expression at various phases of the infection process. Results of these studies will elucidate the basic pathogenic mechanisms involved in GAS disease and may suggest strategies for intervention.